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Platelet-activating-factor—like activity in eosinophils obtained from asthmatic patients
CURRENT THERAPEUTIC RESEARCH” VOL. 56, NO. 5, MAY 1995
PLATELET-ACTIVATING-FACTOR-LIKE ACTIVITY IN EOSINOPHILS OBTAINED FROM ASTHMATIC PATIENTS KUNIHIKO SHINDO AND MOTQNORI FUKUMURA First Department of Intern1 Medicine, Yokohama City University School of Medicine, Yokohama, Japan
ARSTRACT
We investigated platelet-activating-factor (PAF)_like activity in eosinophils obtained from patients with bronchial asthma and from nonasthmatic subjects. The eosinophils were stimulated with formylmethionylleucylphenylalanine (GMLP) (10 pM) for 15 minutes. PAFlike activity was detected via platelet aggregation using washed guinea pig platelets. The PAF-like activity (mean f SD) released from asthmatic eosinophils was 53 f 11% aggregation/lO’ cells in the supernatants and 83 f 15% aggregation/lO’ cells in the cells. PAF-like activity in nonasthmatic eosinophils stimulated with BMLP was 8 f 4% aggregation/lo’ cells in the supematants and 20 f 8% aggregation/ 10’ cells in the cells. Our results demonstrate that PAF-like activity in asthmatic eosinophils is more pronounced than that in nonasthmatic eosinophils, suggesting that asthmatic eosinophils play a role in the development of bronchial asthma through increased PAF-like activity.
INTRODUCTION
Platelet-activating factor (l-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) (PAF) is a potent, biologically active phospholipid that plays a significant role in inflammatory reactions. PAF is synthesized and released by several inflammatory cells, including platelets, neutrophils, macrophages, monocytes, mast cells, eosinophils, and endothelial cells.‘12 However, little is known about the release of PAF from these cells. In particular, there are insufficient data concerning PAF release from asthmatic eosinophils. Human eosinophils are bone marrow-derived polymorphonuclear leukocytes that are thought to contribute to host resistance in some parasitic infections and to modulate the expression of immediate-type hypersensitivity reactions. Eosinophils may play a role in the pathogenesis of
Address correspondence to: Kunihiko Shindo, MD, PhD, 1330-131, Koshigoe, Kamakura 246, Japan. Received for publication on Febmaty 22,1996. printed in the U.S.A. Reproduction in whole or part is not permitted. 486
ooll-393xM/s3.50
K. SHINDO
AND M. FUKUMURA
asthma since eosinophil infiltration is a characteristic feature of asthmatic airways.3 Eosinophil counts in peripheral blood, sputum, and tissue obtained from patients with asthma are closely related to the severity of the disease4-6 and bronchial hyperresponsiveness. 7 Allergen inhalation results in a marked increase in eosinophils in bronchoalveolar lavage fluid at the time of the late reaction.s Eosinophil products have been found at sites of bronchial epithelial damage and denudation in patients with asthma, suggesting a direct involvement of eosinophils in the underlying pathogenic process. Thus the purpose of our experiment was to investigate PAF formation in eosinophils obtained from patients with bronchial asthma. MA'IWRIALSANDMETHODS
Human eosinophils and neutrophils were isolated from the peripheral blood of 18 different donors, none of whom were using aspirin, other nonsteroidal anti-inflammatory drugs, or corticosteroids. Ten donors had atopic-type bronchial asthma; their peripheral eosinophil level (mean + SD) was 12 f 5% (range, 7.1% to 19%). Eosinophils obtained from these donors were considered asthmatic eosinophils. Five donors with no underlying disorders had mean peripheral eosinophil levels of 4.9%, 6.4%, 6.3%, 7.8%, and 7.9%, respectively. Three donors with urticaria had mean eosinophil levels of 9.6%, 10.7%, and 19.6%, respectively. Eosinophils obtained from donors with no underlying disorders and those with urticaria were considered nonasthmatic eosinophils. Informed consent was obtained from all donors. The eosinophils were separated from a mixture of eosinophils and granulocytes by two subsequent centrifugations of a granulocyte preparation in isotonic Percoll solutions (Pharmacia Fine Chemicals, Piscataway, New Jersey) with densities of 1.082 and 1.085 g/mL at 1000 x g for 10 minutes at room temperature. More than 98% pure neutrophils appeared at the top of the 1.082 g/mL Percoll solution, while the eosinophils and some neutrophils were present as a small pellet on the bottom. After resuspension of the eosinophil pellet in a 1.085 g/mL Percoll solution and centrifugation at 1000 x g for 10 minutes at room temperature, more than 90% pure eosinophils appeared at the bottom of the tube. The recovery rate (mean + SD) for eosinophils was 66 ? 13% (n = 18). More than 95% cell viability was confirmed in all experiments by the trypan blue dye exclusion method.gp10 Cells obtained from the asthmatic patients and nonasthmatic donors were washed three times with Hanks’ balanced salt solution without calcium or magnesium and suspended at a density of lo6 cells in 500 ~J,Lof modified Tyrode’s buffer (pH 7.8) containing 0.1% gelatin, 1 mmol calcium, 487
5 mmol magnesium, 0.3 mm01 potassium, and 20 mm01 L-serine. This preparation was then stimulated with formyl-methionylleucylphenylalanine (f-MLP) (10 FM). Reactions were terminated by centrifugation at 12,000 rpm for 1 minute at 22 “C, and the cell pellets and supernatants (media) were frozen at - 70 “C until assayed. PAF-like activity was measured by platelet aggregation using washed guinea pig platelets. Guinea pig platelets were isolated by a modification of the Ardlie’s separation technique as described elsewhere.11212 The change in light transmission through the suspension was monitored for 5 to 10 minutes with an aggregometer. Percent aggregation (ie, change in transmittance) was used to determine the concentration of PAP in each sample. Differences between means were analyzed using a two-tailed unpaired t test. A P value of less than 0.05 was considered significant. Data are expressed as mean + SD.
0
2 A~thmatio Eoeinophm
0
2
8
Nona~thmatio Eosimphils
Figure 1. Representative responses of washed guinea pig platelets to plateletractivating factor in the cell pellets of stiiulated asthmatic and nonasthmatic eosinophils as evaluated by aggregation behavior. These assays were conducted using the change in tranemittance of a platelet suspension.
K.
SHINDO AND M. FUKUMJKA
RESULTS
Representative aggregations are shown in Figure 1. PAF-like activity stimulated by f-MLP was greater in the supematants and cells from the asthmatic eosinophils than from the nonasthmatic eosinophils. The PAFlike activity (mean f SD) in the asthmatic eosinophils was 53 + 11% aggregation/lo’ cells in the supernatants and 83 + 15% aggregation/lO’ cells in the cells (Figure 2). PAF-like activity in nonasthmatic eosinophils stimulated with EMLP was 8 + 4% aggregation/lO’ cells in the supematanta and 20 + 8% aggregation/lO’ cells in the cells. The mean PAP-like activity in the supernatants and cells from stimulated asthmatic eosinophils was significantly higher than that in the supematants and cells from stimulated nonasthmatic eosinophils (P < 0.05). DISCUSSION AND CONCLUSION
Since the early part of this century, physicians have known that bronchial asthma is associated with eosinophilia of the blood and lung.13 Over the past 10 years, several investigators 14-16have published studies on the role of eosinophils in bronchial asthma. However, with the exception of the Lee et al study,” no data are available concerning PAP-like activity in human asthmatic eosinophils. In the report by Lee et all7 eosinophils were isolated from patients with eosinophilia. It is unclear whether these patients had bronchial asthma.
-
A4
p&a
28-c
Figure 2. Platelet-activating-factor-like activity in supematants (8) and cell pellets (c) from formyl-methionylleucylphenylalanine-stimulated asthmatic (A) and nonaethmatic (N) eoeinophile. *P < 0.05, asthmatic vemua nom&lunatic eoeinophils.
PLATELET-ACTIVATING-FACTOR-LJKR
ACTIVITY
IN EOSJNOPHILS
Fukuda et al” reported that a portion of eosinophils from asthmatic patients and most eosinophils from patients with hypereosinophilic syndrome are hypodense. This finding may reflect the release of immature eosinophils from bone marrow or the activation of eosinophils in the peripheral circulation, although the true reason for this hypodensity of eosinophils is unknown. The increased PAF-like activity seen in asthmatic eosinophils in our study may reflect the activation of hypodense eosinophils in the peripheral circulation. Our data demonstrate that f-MLP-stimulated PAF-like activity in supernatants and cells from human asthmatic eosinophils was significantly higher than that in nonasthmatic eosinophils. Other investigators have shown that asthmatic eosinophils also release more leukotriene C4” and major basic protein 2o than nonasthmatic eosinophils. Thus asthmatic eosinophils may play a role in the development of bronchial asthma through increased PAF-like activity.
Acknowledgment The authors gratefully acknowledge Dr. M. Tanaka for reviewing this manuscript. References: 1. Braquet P, Shen TV, VergraRig BB. Perspectives in platelet-activating factor research. Phrmacol Rev. 1987;39:127-139. 2. Barnes PJ, Chung KF, Page CP. Platelet activating factor as a mediator in allergic disease. J Allergy Clin Immud. 1988;81:919-929. 3. Filley WV, Holley KE, Kephart GM, Gleich GJ. Identification by immunofluorescence of eosinophil granule major basic protein in lung tissue of patients with bronchial asthma. Luncet. 1982;2:11-15. 4. Burrows B, Hasan FM, Barbee RM, et al. Epidemiological observations on eosinophilia and its relation to respiratory disease. Am Rev Respir Dia. 1980;122:709-718. 5. Taylor J, Luksza AR. Peripheral blood eosinophil counts and bronchial responsiveness. Thorax. 1987;42:452-461. 6. Wardlaw AJ, Dunette S, Gleich GJ, et al. Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma: Relationship to bronchial hyperreactivity. Am Rev Respir Dis. 1988;137:62-70. 7. Frigas E, Gleich GJ. The eosinophil and the pathophysiology of asthma. J Allergy Clin Immurwl. 1986;77:527-538. 8. DeMonchy JGR, Kauffman HF, Venge P, et al. Bronchoalveolar eosinophils during allergen-induced late asthmatic reactions. Am Rev Respir Dis. 1985;131:373-388. 9. Verhagen J, Bruynzeel PLB, Koedam JA, et al. Leukotriene formation by purified human eosinophils and neutrophils. Febs Lett. 1984,168:23-28. 490
K.SHINDO ANDM.FUKUMURA
10. Bruynzeel PLB, Kok PTM, Vietor RJ, Verhagen J. On the optimal conditions of LTC4 formation by human eoeinophils in vitro. Prostaglandins Leukot Med. 1985;20:11-22. 11. Ardlie NG, Packham MA, Mustard JF. Adenosine diphosphate induced platelet aggregation in suspensions of washed rabbit platelets. Br J Haemntol. 1970;19:7-16. 12. Pinckard RN, Farr RN, Hanahan DJ. Physicochemical and functional identity of rabbit platelet-activating factor (PAF) released in vivo during IgE anaphylaxis with PAF released in vitro from IgE sensitized basophils. J Zmmunol. 1979;123:1847-1857. 13. Ellis AG. The pathological anatomy of bronchial asthma. Am J Med Sci. 1980;136:407417. 14. Jorg A, Henderson WR, Murphy RC, Klebanoff SJ. Leukotriene generation by eosinophils. J Ezp Med. 1982;155:390-402. 15. Henderson WR, Chi EY, Klebanoff SJ. Eosinophil peroxidase-induced mast cell secretion. J Exp Med. 1980;152:265-279. 16. Weinstock JV, Blum A, Walder J, Walder R. Eosinophils from granulomas in murine Schistosomiasis mansoni produced substance P. J Zmmunol. 1988;141:961-966. 17. Lee T, Lenihan DJ, Malone B, et al. Increased biosynthesis of platelet-activating factor in activated human eosinophils. J Biol Chem. 1984;259:5526-5530. 18. Fukuda T, Dunnette SL, Reed CE, et al. Increased numbers of hypodense eosinophils in the blood of patients with bronchial asthma. Am Rev Respir Dis. 1985;132:981-985. 19. Bousquet J, Chanez P, Vignola AM, et al. Eosinophil inflammation in asthma. Am J Respir Crit Care Med. 1994;15O(Suppl):533-538. 20. O’Donnell MC, Ackerman SJ, Gleich GJ, Thomas LL. Activation ofbasophil and mast cell histamine release by eosinophil granule major basic protein. JExp Med. 1983;157:19811991.
491
PLATELET-ACTIVATING-FACTOR-LIKE ACTIVITY IN EOSINOPHILS OBTAINED FROM ASTHMATIC PATIENTS KUNIHIKO SHINDO AND MOTQNORI FUKUMURA First Department of Intern1 Medicine, Yokohama City University School of Medicine, Yokohama, Japan
ARSTRACT
We investigated platelet-activating-factor (PAF)_like activity in eosinophils obtained from patients with bronchial asthma and from nonasthmatic subjects. The eosinophils were stimulated with formylmethionylleucylphenylalanine (GMLP) (10 pM) for 15 minutes. PAFlike activity was detected via platelet aggregation using washed guinea pig platelets. The PAF-like activity (mean f SD) released from asthmatic eosinophils was 53 f 11% aggregation/lO’ cells in the supernatants and 83 f 15% aggregation/lO’ cells in the cells. PAF-like activity in nonasthmatic eosinophils stimulated with BMLP was 8 f 4% aggregation/lo’ cells in the supematants and 20 f 8% aggregation/ 10’ cells in the cells. Our results demonstrate that PAF-like activity in asthmatic eosinophils is more pronounced than that in nonasthmatic eosinophils, suggesting that asthmatic eosinophils play a role in the development of bronchial asthma through increased PAF-like activity.
INTRODUCTION
Platelet-activating factor (l-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) (PAF) is a potent, biologically active phospholipid that plays a significant role in inflammatory reactions. PAF is synthesized and released by several inflammatory cells, including platelets, neutrophils, macrophages, monocytes, mast cells, eosinophils, and endothelial cells.‘12 However, little is known about the release of PAF from these cells. In particular, there are insufficient data concerning PAF release from asthmatic eosinophils. Human eosinophils are bone marrow-derived polymorphonuclear leukocytes that are thought to contribute to host resistance in some parasitic infections and to modulate the expression of immediate-type hypersensitivity reactions. Eosinophils may play a role in the pathogenesis of
Address correspondence to: Kunihiko Shindo, MD, PhD, 1330-131, Koshigoe, Kamakura 246, Japan. Received for publication on Febmaty 22,1996. printed in the U.S.A. Reproduction in whole or part is not permitted. 486
ooll-393xM/s3.50
K. SHINDO
AND M. FUKUMURA
asthma since eosinophil infiltration is a characteristic feature of asthmatic airways.3 Eosinophil counts in peripheral blood, sputum, and tissue obtained from patients with asthma are closely related to the severity of the disease4-6 and bronchial hyperresponsiveness. 7 Allergen inhalation results in a marked increase in eosinophils in bronchoalveolar lavage fluid at the time of the late reaction.s Eosinophil products have been found at sites of bronchial epithelial damage and denudation in patients with asthma, suggesting a direct involvement of eosinophils in the underlying pathogenic process. Thus the purpose of our experiment was to investigate PAF formation in eosinophils obtained from patients with bronchial asthma. MA'IWRIALSANDMETHODS
Human eosinophils and neutrophils were isolated from the peripheral blood of 18 different donors, none of whom were using aspirin, other nonsteroidal anti-inflammatory drugs, or corticosteroids. Ten donors had atopic-type bronchial asthma; their peripheral eosinophil level (mean + SD) was 12 f 5% (range, 7.1% to 19%). Eosinophils obtained from these donors were considered asthmatic eosinophils. Five donors with no underlying disorders had mean peripheral eosinophil levels of 4.9%, 6.4%, 6.3%, 7.8%, and 7.9%, respectively. Three donors with urticaria had mean eosinophil levels of 9.6%, 10.7%, and 19.6%, respectively. Eosinophils obtained from donors with no underlying disorders and those with urticaria were considered nonasthmatic eosinophils. Informed consent was obtained from all donors. The eosinophils were separated from a mixture of eosinophils and granulocytes by two subsequent centrifugations of a granulocyte preparation in isotonic Percoll solutions (Pharmacia Fine Chemicals, Piscataway, New Jersey) with densities of 1.082 and 1.085 g/mL at 1000 x g for 10 minutes at room temperature. More than 98% pure neutrophils appeared at the top of the 1.082 g/mL Percoll solution, while the eosinophils and some neutrophils were present as a small pellet on the bottom. After resuspension of the eosinophil pellet in a 1.085 g/mL Percoll solution and centrifugation at 1000 x g for 10 minutes at room temperature, more than 90% pure eosinophils appeared at the bottom of the tube. The recovery rate (mean + SD) for eosinophils was 66 ? 13% (n = 18). More than 95% cell viability was confirmed in all experiments by the trypan blue dye exclusion method.gp10 Cells obtained from the asthmatic patients and nonasthmatic donors were washed three times with Hanks’ balanced salt solution without calcium or magnesium and suspended at a density of lo6 cells in 500 ~J,Lof modified Tyrode’s buffer (pH 7.8) containing 0.1% gelatin, 1 mmol calcium, 487
5 mmol magnesium, 0.3 mm01 potassium, and 20 mm01 L-serine. This preparation was then stimulated with formyl-methionylleucylphenylalanine (f-MLP) (10 FM). Reactions were terminated by centrifugation at 12,000 rpm for 1 minute at 22 “C, and the cell pellets and supernatants (media) were frozen at - 70 “C until assayed. PAF-like activity was measured by platelet aggregation using washed guinea pig platelets. Guinea pig platelets were isolated by a modification of the Ardlie’s separation technique as described elsewhere.11212 The change in light transmission through the suspension was monitored for 5 to 10 minutes with an aggregometer. Percent aggregation (ie, change in transmittance) was used to determine the concentration of PAP in each sample. Differences between means were analyzed using a two-tailed unpaired t test. A P value of less than 0.05 was considered significant. Data are expressed as mean + SD.
0
2 A~thmatio Eoeinophm
0
2
8
Nona~thmatio Eosimphils
Figure 1. Representative responses of washed guinea pig platelets to plateletractivating factor in the cell pellets of stiiulated asthmatic and nonasthmatic eosinophils as evaluated by aggregation behavior. These assays were conducted using the change in tranemittance of a platelet suspension.
K.
SHINDO AND M. FUKUMJKA
RESULTS
Representative aggregations are shown in Figure 1. PAF-like activity stimulated by f-MLP was greater in the supematants and cells from the asthmatic eosinophils than from the nonasthmatic eosinophils. The PAFlike activity (mean f SD) in the asthmatic eosinophils was 53 + 11% aggregation/lo’ cells in the supernatants and 83 + 15% aggregation/lO’ cells in the cells (Figure 2). PAF-like activity in nonasthmatic eosinophils stimulated with EMLP was 8 + 4% aggregation/lO’ cells in the supematanta and 20 + 8% aggregation/lO’ cells in the cells. The mean PAP-like activity in the supernatants and cells from stimulated asthmatic eosinophils was significantly higher than that in the supematants and cells from stimulated nonasthmatic eosinophils (P < 0.05). DISCUSSION AND CONCLUSION
Since the early part of this century, physicians have known that bronchial asthma is associated with eosinophilia of the blood and lung.13 Over the past 10 years, several investigators 14-16have published studies on the role of eosinophils in bronchial asthma. However, with the exception of the Lee et al study,” no data are available concerning PAP-like activity in human asthmatic eosinophils. In the report by Lee et all7 eosinophils were isolated from patients with eosinophilia. It is unclear whether these patients had bronchial asthma.
-
A4
p&a
28-c
Figure 2. Platelet-activating-factor-like activity in supematants (8) and cell pellets (c) from formyl-methionylleucylphenylalanine-stimulated asthmatic (A) and nonaethmatic (N) eoeinophile. *P < 0.05, asthmatic vemua nom&lunatic eoeinophils.
PLATELET-ACTIVATING-FACTOR-LJKR
ACTIVITY
IN EOSJNOPHILS
Fukuda et al” reported that a portion of eosinophils from asthmatic patients and most eosinophils from patients with hypereosinophilic syndrome are hypodense. This finding may reflect the release of immature eosinophils from bone marrow or the activation of eosinophils in the peripheral circulation, although the true reason for this hypodensity of eosinophils is unknown. The increased PAF-like activity seen in asthmatic eosinophils in our study may reflect the activation of hypodense eosinophils in the peripheral circulation. Our data demonstrate that f-MLP-stimulated PAF-like activity in supernatants and cells from human asthmatic eosinophils was significantly higher than that in nonasthmatic eosinophils. Other investigators have shown that asthmatic eosinophils also release more leukotriene C4” and major basic protein 2o than nonasthmatic eosinophils. Thus asthmatic eosinophils may play a role in the development of bronchial asthma through increased PAF-like activity.
Acknowledgment The authors gratefully acknowledge Dr. M. Tanaka for reviewing this manuscript. References: 1. Braquet P, Shen TV, VergraRig BB. Perspectives in platelet-activating factor research. Phrmacol Rev. 1987;39:127-139. 2. Barnes PJ, Chung KF, Page CP. Platelet activating factor as a mediator in allergic disease. J Allergy Clin Immud. 1988;81:919-929. 3. Filley WV, Holley KE, Kephart GM, Gleich GJ. Identification by immunofluorescence of eosinophil granule major basic protein in lung tissue of patients with bronchial asthma. Luncet. 1982;2:11-15. 4. Burrows B, Hasan FM, Barbee RM, et al. Epidemiological observations on eosinophilia and its relation to respiratory disease. Am Rev Respir Dia. 1980;122:709-718. 5. Taylor J, Luksza AR. Peripheral blood eosinophil counts and bronchial responsiveness. Thorax. 1987;42:452-461. 6. Wardlaw AJ, Dunette S, Gleich GJ, et al. Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma: Relationship to bronchial hyperreactivity. Am Rev Respir Dis. 1988;137:62-70. 7. Frigas E, Gleich GJ. The eosinophil and the pathophysiology of asthma. J Allergy Clin Immurwl. 1986;77:527-538. 8. DeMonchy JGR, Kauffman HF, Venge P, et al. Bronchoalveolar eosinophils during allergen-induced late asthmatic reactions. Am Rev Respir Dis. 1985;131:373-388. 9. Verhagen J, Bruynzeel PLB, Koedam JA, et al. Leukotriene formation by purified human eosinophils and neutrophils. Febs Lett. 1984,168:23-28. 490
K.SHINDO ANDM.FUKUMURA
10. Bruynzeel PLB, Kok PTM, Vietor RJ, Verhagen J. On the optimal conditions of LTC4 formation by human eoeinophils in vitro. Prostaglandins Leukot Med. 1985;20:11-22. 11. Ardlie NG, Packham MA, Mustard JF. Adenosine diphosphate induced platelet aggregation in suspensions of washed rabbit platelets. Br J Haemntol. 1970;19:7-16. 12. Pinckard RN, Farr RN, Hanahan DJ. Physicochemical and functional identity of rabbit platelet-activating factor (PAF) released in vivo during IgE anaphylaxis with PAF released in vitro from IgE sensitized basophils. J Zmmunol. 1979;123:1847-1857. 13. Ellis AG. The pathological anatomy of bronchial asthma. Am J Med Sci. 1980;136:407417. 14. Jorg A, Henderson WR, Murphy RC, Klebanoff SJ. Leukotriene generation by eosinophils. J Ezp Med. 1982;155:390-402. 15. Henderson WR, Chi EY, Klebanoff SJ. Eosinophil peroxidase-induced mast cell secretion. J Exp Med. 1980;152:265-279. 16. Weinstock JV, Blum A, Walder J, Walder R. Eosinophils from granulomas in murine Schistosomiasis mansoni produced substance P. J Zmmunol. 1988;141:961-966. 17. Lee T, Lenihan DJ, Malone B, et al. Increased biosynthesis of platelet-activating factor in activated human eosinophils. J Biol Chem. 1984;259:5526-5530. 18. Fukuda T, Dunnette SL, Reed CE, et al. Increased numbers of hypodense eosinophils in the blood of patients with bronchial asthma. Am Rev Respir Dis. 1985;132:981-985. 19. Bousquet J, Chanez P, Vignola AM, et al. Eosinophil inflammation in asthma. Am J Respir Crit Care Med. 1994;15O(Suppl):533-538. 20. O’Donnell MC, Ackerman SJ, Gleich GJ, Thomas LL. Activation ofbasophil and mast cell histamine release by eosinophil granule major basic protein. JExp Med. 1983;157:19811991.
491